Evaluation of the risk of foreign compound exposure depends upon a knowledge of xenobiotic metabolism. This proposal targets two enzyme classes, epoxide hydrolases (EHs) and esterases, which are both members of the alpha/beta-hydrolase fold family. Rodent and human EHs and esterases expressed in the baculovirus system are used to develop an understanding of their catalytic mechanism based on a variety of tools including X-ray crystallography, homology modeling, enzyme kinetics, synthesis of enzyme chimera, and site directed mutagenesis. An immediate benefit is the ability to screen human hydrolases for possible inhibitors while a long term goal is development of a mechanistic basis to predict substrate and inhibitor binding. Highly toxic epoxides are hydrolyzed in part by EHs. The structural information gained in objective 1 advances understanding enzyme catalysis and inhibition by several compounds including commercial herbicides and insecticides. The message and gene are being used to determine the basis of the unique dual localization of the sEH in mammalian cells. Objective II addresses the hypothesis that changes in EH activity influence health by changing the metabolic profile of endogenous, biologically active fatty acid epoxides. The hypothesis that biologically activity attributed to epoxides of linoleic acid actually is due to generation of a toxic diol metabolite by the sEH will be tested. Analytic methods for the epoxides, diols and conjugates in serum and urine are being developed based on ion trap GLC-MS and immunoassay. Toxicity and metabolism of these oxidized lipids (oxylipins) are examined in transgenic cells expressing key enzymes, in target cells such as alveolar epithelium and resistant cells such as hepatocytes. These studies will be extended in vivo for evaluating interaction of xenobiotics with oxylipin toxicity. Finally, the hypothesis that oxylipin rations in exposed workers are altered by xenobiotic exposure by monitoring oxylipins and herbicide inhibitors of EH urines will be tested. Inhibition of esterases is a well established cause of toxicity. The esterases primarily responsible for the hydrolysis of the insecticides permethrin and malathion are being purified, cloned and expressed in objective III. The recombinant enzymes will be used for developing a structural basis for predicting inhibitors of these enzymes and thus combinations of insecticides which present toxic risk. For both EHs and esterases the hypothesis of a catalytic tetrad in a two step mechanism will be tested.
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